Search Results (92)

Background/Objective: Leishmaniasis is a neglected tropical disease caused by species of the genus Leishmania, with a broad clinical spectrum that can overlap with other infectious and non-infectious conditions. Accurate species identification is critical for appropriate treatment and prognosis; however, parasitological methods are limited by suboptimal sensitivity, specificity, and inability to reliably differentiate species. This study aimed to develop and validate a real-time PCR assay based on melting-curve analysis (Leish-qPCR) for the detection of Leishmania spp. and the differentiation of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. Methods and Results: Genus-specific primers were designed based on the kDNA (kinetoplast DNA) minicircle consensus sequences of Leishmania species, while species-specific primers targeted the internal transcribed spacer 2 (ITS2) consensus regions of the ribosomal RNA locus of L. (L.) amazonensis and L. (V.) braziliensis. Analytical performance was evaluated in silico and in vitro using a panel of protozoa, fungi, and bacteria, exhibiting 100% specificity with no cross-amplification. The limit of detection was one copy per reaction for all targets using positive controls. Clinical validation was performed using skin biopsy specimens from patients with granulomatous lesions. The optimized Leish-qPCR assay, performed in separate reaction tubes within the same run, demonstrated reliable analytical specificity and sensitivity, with distinct and reproducible melting temperature (Tm) peaks across plasmid controls, parasite DNA, and clinical samples. Comparative analysis with histopathological examination demonstrated moderate agreement between the methods, supporting the applicability of the assay for sensitive detection and species-level discrimination of Leishmania spp. in clinical samples. Conclusions: The Leish-qPCR assay presented high sensitivity, specificity, and diagnostic accuracy, representing a promising tool for routine diagnosis of leishmaniasis and for the differentiation of L. (L.) amazonensis and L. (V.) braziliensis in clinical samples. Full article

An outbreak investigation of camelpox in dromedary camels (Camelus dromedarius) was conducted from October 2024 to May 2025 in Borena Zone, Ethiopia, with the aims of isolating, confirming the etiology and molecular characterization of camelpox virus from outbreak cases. The study integrated clinical assessment, virus isolation using Vero cell lines, and molecular characterization using conventional PCR, real-time PCR, sequencing and phylogenetic analysis. Clinically affected camels manifested typical pox-like lesions, fever and swollen lymph nodes, with a morbidity rate of 33.8% (24/71) and a case fatality rate of 4.2% (1/24). The virus was successfully isolated showing typical cytopathic effects of rounding of cells, syncytia, giant cell formation, aggregation and detachment. Conventional PCR targeting A-type inclusion protein (ATIP) gene amplified the expected 881 bp fragment with 26.3% positivity in both skin scab and nasal swab samples. Real-time PCR employing high-resolution melting curve analysis detected the viral DNA in 52.6% of samples with a melting temperature of 73.00 ± 0.20 °C for CMPV. Sequencing of the ATIP gene showed 100% nucleotide similarity with reference CMPV strains of CMPV M-96, CMPV CMS, strain 0408151v and CMPV genome (NC_003391), although a single nucleotide variation was noted when compared to the previously reported Ethiopian isolates (KU705085-KU705110) and Israeli isolates (MK910851 and MZ300856), and two nucleotide mismatches were observed with Sudanese isolates (KT931624 and KT931625). Phylogenetic analysis revealed that the current isolates clustered with CMPV strains of CMPV M-96, CMPV CMS, strain 0408151v and others but were distinct from previously reported Ethiopian isolates. This study provides significant insights on early diagnosis and control strategies. Full article

In this study, a SYBR Green-based real-time PCR workflow targeting the vdcC gene was optimized and validated for rapid detection of Alicyclobacillus acidoterrestris in fruit juice. A commercial DNA extraction kit showed the best performance, achieving a limit of detection (LOD) of 2 Log CFU/mL, whereas microwave-based extraction (30 s) provided a rapid alternative with an LOD of 3 Log CFU/mL. The vdcC primer set enabled clear discrimination of A. acidoterrestris from closely related species based on melting curve analysis. The method was successfully applied to orange, apple, and grape juice matrices, as well as to 30 commercial juice samples. Guaiacol analysis further indicated that the presence of A. acidoterrestris DNA did not necessarily correspond to active spoilage. Overall, this study provides a systematically optimized and practically validated workflow for monitoring A. acidoterrestris in fruit juice systems. Full article

Singleplex and multiplex real-time PCR-HRM (polymerase chain reaction with high resolution melting), both with specific and non-specific amplicon detection, are used for a wide range of applications, from clinical diagnostics to food authentication. However, their results can be influenced by the quality of the template DNA and composition of the reaction mixture. The methods used for the analysis of these results then influence the conclusions drawn. In this work we present an example from our laboratory practice, where the results of singleplex and multiplex real-time PCR differed, despite using the same reaction conditions, primers and analyzed plant material. We show the influence of a singleplex and multiplex PCR setup on the results, as well as the influence of template contamination on the melting behaviour of amplicons. We also discuss the usefulness of cluster analysis for the clarification of real-time PCR-HRM results which appear unclear when only melting and difference curves or similarity scores are used for the analysis of these results. We provide a discussion of problems which we encountered during an analysis of commercial teas and which should be considered by researchers new to PCR-based analysis of plant material, especially if the studied material is rich in various contaminants. Full article

A liver fluke, Clonorchis sinensis is a representative fish-borne parasite infecting humans, and sensitive detection in fish hosts or aquatic environments is important for monitoring infection sources in endemic areas. Conventional diagnostic methods based on microscopy or conventional PCR often show limited sensitivity, particularly under low-parasite conditions. In this study, we developed a high-sensitive and species-specific molecular marker and established a real-time PCR (qPCR)-based diagnostic method targeting metacercariae isolated from freshwater fish, representing the transmission stage of C. sinensis. Primers and a hydrolysis probe targeting the mitochondrially encoded cytochrome c oxidase 1 (COI) gene were designed, and all primer combinations produced stable amplifications with single melt curves in C. sinensis-positive samples. Among them, one combination was finally selected as the optimal marker due to its high specificity, including validation against mixed trematode samples to confirm species-specific detection. The qPCR assay showed excellent linearity (R² = 0.998), with a detection limit of 10¹ copies per reaction and a quantification limit of 10² copies per reaction. In addition, the assay successfully detected C. sinensis DNA in environmental water samples spiked with metacercariae, demonstrating its applicability to aquatic samples for environmental surveillance purposes. Compared with conventional PCR, the developed qPCR method in this study exhibited markedly improved sensitivity in fish-derived samples. Overall, this qPCR assay provides a robust diagnostic tool for laboratory analysis and has potential utility for environmental DNA-based monitoring of clonorchiasis risk areas within a One Health framework. Full article

Purslane (Portulaca oleracea L.) is an important plant species that has been increasingly used in functional gene studies and molecular analyses. However, reference genes that exhibit stable expression across multiple tissues and stress conditions have not been systematically validated in purslane, which limits the accuracy of reverse transcription quantitative PCR (RT-qPCR) based gene expression analyses. In this study, ten candidate reference genes from six gene families (Actin, PP2A, CYP, eIF4A, Ubiquitin, and eIF5A) were selected based on transcriptome data. A combination of bioinformatic analyses and experimental validation was employed to comprehensively characterize these candidates, including their physicochemical properties, chromosomal localization, phylogenetic relationships, gene structures, and promoter cis-acting elements. Furthermore, the expression stability of the candidate genes was systematically evaluated across different tissues (seed, root, stem, leaf, and flower) and under multiple stress treatments, including salinity, temperature stress, drought, and hormone treatments. Based on conventional PCR amplification specificity, melting curve analysis, Ct value distribution, and amplification efficiency, ACT-2 and eIF5A-1 were identified as the most stably expressed reference genes under diverse experimental conditions. This study provides reliable reference gene candidates for accurate normalization of gene expression in purslane and establishes a systematic framework for reference gene selection in non-model plant species. Full article

Molecular sex determination in Syrian hamsters (Mesocricetus auratus) has been limited by the incomplete annotation of Y-linked loci in currently available genome assemblies. Here, we evaluate the Y-linked gene PRSSLY, which encodes a testis-specific serine protease-like protein, as a molecular marker for genetic sexing of Syrian hamster embryonic and placental tissues. Primers flanking a conserved PRSSLY coding region produced a male-specific amplicon showing 100% concordance with results from the established KDM5C/KDM5D PCR assay in E15.5 tail biopsies. SYBR Green–based qPCR enables the accurate detection of PRSSLY, characterized by a unique melt-curve profile, exclusively in male samples, allowing for efficient and sensitive mid-throughput analysis. Application of the PRSSLY assay to 417 placental samples from 39 dams demonstrated its suitability for large-scale sex genotyping, enabling sex assignment in the majority of samples despite the intrinsic complexity of placental tissue containing both maternal and embryonic genetic material. This assay provides a robust and reproducible approach for accurate sex genotyping in developmental and reproductive studies using Syrian hamsters. Full article

Heterocapsa bohaiensis is an emerging harmful dinoflagellate increasingly reported from coastal regions of the Pacific. However, an available molecular assay offering rapid and sensitive detection is still lacking. This study developed a SYBR Green real-time quantitative PCR (qPCR) assay for the identification and quantification of H. bohaiensis. Species-specific primers (F: 5′-CCATCGAACCAGAACTCCGT-3′; R: 5′-AGTGTAGTGCACCGCATGTC-3′) were designed and the assay was optimized and evaluated using laboratory cultures for specificity, sensitivity, and quantitative performance. Primer screening and melt-curve analysis confirmed that the selected primer pair produced a single, specific amplification peak for H. bohaiensis, with no cross-reactivity observed in non-target species (Chlorella pyrenoidosa, Phaeocystis globosa, Skeletonema costatum, Alexandrium tamarense) or mixed algal communities. The standard curve displayed strong linearity (R² = 0.9868) and a high amplification efficiency (102.5%). The limit of detection (LOD) was approximately 2–3 cells per reaction, as determined from 24 replicates of 5-cell equivalents and verified at ~2.7-cell equivalents. This sensitivity was comparable to or exceeded that reported for assays targeting other HABs forming dinoflagellates. Quantitative results derived from the qPCR assay closely matched microscopic cell counts, with a relative error of 10.79%, falling within the acceptable threshold for phytoplankton surveys. In summary, this study established and validates a species-specific qPCR assay for H. bohaiensis under controlled laboratory conditions. The method shows strong potential for incorporation into HAB monitoring programs, early-warning systems, and future ecological investigations of this emerging species. Full article

In this study, we focused on the screening and identification of reference genes for Paracarophenax alternatus Xu and Zhang. The laboratory population was used as the laboratory population, and samples were collected from mites at four different stages, including physogastry, viviparous, 5 d viviparous and phoresy. Then, the expression levels of seven candidate reference genes (α-tubulin, β-tubulin, RPS18, RPL13, GAPDH, EF1A, SDHA) were detected through qRT-PCR. Melting curves showed good gene specificity, and the amplification efficiency ranged from 90% to 102%. ΔCt analysis indicated that GAPDH was the most stable reference gene. The GeNorm software determined that the optimal number of reference genes was two, with GAPDH and RPS18 forming the most stable combination, and NormFinder identified RPS18 as the most stable reference gene. Although the BestKeeper software suggested that EF1A was the most stable, its p-value exceeded 0.05, rendering it unsuitable for use as a reference gene. Finally, through the RefFinder network tool, the most stable reference genes were identified as GAPDH and RPS18. Full article

Background: The cytochrome P450 family 21 subfamily A member 2 gene (CYP21A2) encodes 21-hydroxylase, a key enzyme in adrenal steroid biosynthesis. Despite its physiological importance, the diversity of CYP21A2 transcript variants and their tissue-specific expression in domestic animals, including cattle, remains largely unexplored. This study aimed to characterize CYP21A2 transcription in adrenal glands and ovaries and assess the potential transcriptional activity of its pseudogene, CYP21A1P. Methods: CYP21A2 transcription was investigated in adrenal and ovarian tissues of 12 healthy cows using semi-quantitative PCR and Sanger sequencing. Real-time PCR was performed to confirm expression levels. Melting curve analysis and electrophoresis were used to validate distinct amplicons corresponding to different transcript variants. Extended amplicons were sequenced to identify transcripts corresponding to reference sequences and potential pseudogene products. Results: A single transcript variant (NM_001013596.1) was consistently detected in adrenal glands, whereas ovaries expressed two variants: NM_001013596.1 and XM_024983378.2. Semi-quantitative analysis showed significantly higher CYP21A2 expression in adrenal glands compared to ovaries (p < 0.01). In ovarian samples, the NM_001013596.1 variant was more abundant than the XM_024983378.2 (p < 0.01). Sanger sequencing revealed two products matching CYP21A2 reference transcripts and an additional, longer product containing sequence motifs specific to the pseudogene CYP21A1P, indicating its transcriptional activity. Conclusions: These results provide the first evidence of tissue-specific expression and differential abundance of CYP21A2 transcript variants in cattle and suggest the transcription of the CYP21A1P pseudogene. The findings reveal the complexity of CYP21A2 expression in steroidogenic tissues and suggest potential regulatory roles for transcript and pseudogene variants in bovine physiology. Full article

Background and Objectives: Thrombophilia is a prothrombotic disorder that increases the risk of blood clotting and can pose serious health problems. It is considered a condition of gene–gene or gene–environment interactions. Variation in the prevalence of thrombophilia mutations and their interaction among populations necessitates localized genetic assessments. However, population-based genetic data remains limited for developing effective preventive strategies. Materials and Methods: This cross-sectional observational study was conducted over five years (2020–2024) at a tertiary university hospital in Northern Greece. A total of 2961 individuals aged 18–85 years (mean: 50.5) were registered based on family or medical history of venous thromboembolism (VTE) or clinical symptoms of VTE. The final analysis included 2078 participants comprising 1143 males (55%) and 935 females (45%), who met all the inclusion criteria. Inclusion criteria were absence of acute illness or malignancy, informed consent, and an adequate DNA quantity for genotyping, whereas excluded criteria included incomplete laboratory data, active inflammatory or malignant disease, and cognitive or psychiatric conditions. Peripheral blood samples were collected in 2 mL K₃-EDTA tubes, and genomic DNA was analyzed using real-time polymerase chain reaction (PCR) with melting curve analysis and hybridization probes (LightMix^® in vitro diagnostics, TIB MolBiol, Berlin, Germany). Five thrombophilia-related polymorphisms, Factor V Leiden (F5 G1691A), prothrombin (F2 G20210A), methylenetetrahydrofolate reductase (MTHFR C677T and MTHFR A1298C), and Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G, were examined for allele and genotype frequencies, Hardy–Weinberg equilibrium testing, pairwise linkage disequilibrium (D′ and r²), and power analysis. For subjects tested for Factor V Leiden (n = 1476), the activated protein C resistance (APC) ratio was additionally evaluated using the ACL TOP 750 analyzer. Results: Allele frequencies were 7.3% for FV Leiden and 3.7% for FII. The PAI-1 allele was distributed at 44%, while the MTHFR (C677T and A1298C) alleles were each present at 33%. Significant linkage disequilibrium was identified between MTHFR (C677T and A1298C) and between MTHFR A1298C and PAI-1. No evolutionary pressure or demographic bias was found in the Hardy–Weinberg equilibrium. The APC ratio demonstrated a high sensitivity (99.2%) and specificity (96.6%), indicating that it may serve as a reliable screening method. Conclusions: Our findings highlight informative patterns in the genetic predisposition to thrombophilia, which may help develop rule-based strategies for implementing thromboprophylaxis guidelines and personalized medical interventions. Full article

Carotid artery intima–media thickness (cIMT), a pre-clinical vascular change that accompanies atherosclerosis is considered as a cardiovascular risk factor. Coagulation factor XIII (FXIII) stabilizes the fibrin clot and increases its resistance to fibrinolysis. Regarding FXIII Val34Leu polymorphism, the protective effect of the Leu34 allele in the presence of elevated fibrinogen levels against myocardial infarction was demonstrated. Our aim was to investigate the effect of FXIII polymorphisms on cIMT. Patients with obesity (n = 69), type 2 diabetes mellitus (T2DM) (n = 104), and age- and sex-matched healthy controls (n = 82) were enrolled. FXIII polymorphisms (Val34Leu, His95Arg, Intron-K C>G) were determined by RT-PCR with FRET detection and melting curve analysis. cIMT was determined by B-mode ultrasound. Differences in cIMT between control (median: 0.5965, IQR: 0.5115–0.6580 mm) and T2DM (median: 0.7105, IQR: 0.5948–0.7568 mm), as well as between obese (median: 0.6105, IQR: 0.5455–0.6780 mm) and diabetic groups, were found (p < 0.0001 and p = 0.003, respectively). Genotype and allele frequencies of the studied polymorphisms did not differ between subgroups. In the study group (n = 255) after adjustment for age and sex, the presence of Intron-K G allele showed a significant and independent protective effect against cIMT progression in a separate model (p = 0.005) and after adjusting for other parameters associated with cIMT (p = 0.015). FXIII Intron-K G allele provides a protective effect against subclinical vascular disease in the studied population, and this effect is independent of the presence of obesity, as well as T2DM, Leu34 allele, and fibrinogen levels. Full article

Salix suchowensis is an ideal model organism for investigating nitrogen (N) transport mechanisms due to its low N-input requirements. Accurate quantification of gene expression is essential for elucidating these processes, with quantitative real-time PCR (RT-qPCR) being the preferred method. However, the identification of stable reference genes for normalization in Salix suchowensis under varying N conditions remains unresolved. In this study, thirteen commonly employed candidate reference genes were evaluated across root, stem, and leaf tissues, under four N treatments (NH₄NO₃, NH₄⁺, NO₃⁻, and N deficiency). Five genes (UBQ1, UBQ3, 18S, H2A2, and H2B2) were excluded due to poor amplification efficiency or irregular melting curves. The remaining eight genes were further assessed for expression stability using the geNorm, NormFinder, and BestKeeper algorithms. Integrated ranking via RefFinder identified EF1α, EFβ, and αTUB as the most stable reference genes. GeNorm analysis suggested that two reference genes were sufficient for reliable normalization. Validation using the N-responsive gene SsAMT1 and SsNRT2 confirmed the stability of EF1α, EFβ, and αTUB as suitable reference genes. Based on comprehensive stability assessments and experimental validation, we recommended EF1α + αTUB as optimal reference gene pairs for RT-qPCR normalization under varying N conditions. Furthermore, the consistent expression of EF1α and αTUB across nine willow genotypes highlighted their broader applicability within Salix species. This study provides valuable methodological guidance for advancing molecular research on N transport in woody perennial plants. Full article

Background/Objectives: Phosphoglucomutase-1 (PGM1) is an enzyme that plays important roles in glycolysis, glycogen metabolism, and glycosylation. The PGM1 gene harbors two common nonsynonymous single-nucleotide variants (rs1126728 and rs11208257), which result in four functional PGM1 phenotypes. Correlations between PGM1 polymorphisms and several pathological conditions have been suggested. Methods: To identify the rs1126728 and rs11208257 concurrently, a fluorescence melting curve analysis (FMCA) was developed that utilizes two distinct dual-labeled fluorescence probes. Two distinct Taq polymerases, one with and one without 5′-3′exonuclease activity, were compared. This method was then applied to 95 unrelated Japanese subjects. Results: Both Taq polymerases, with and without 5′-3′exonuclease activity, were found to be sufficiently functional. Furthermore, the results of the FMCA using both Taq polymerases were compared with the direct Sanger sequencing results of PCR products from the 95 Japanese subjects, demonstrating 100% concordance. Conclusions: The duplex probe-based FMCA developed in this study is useful for examining the association between rs1126728 or rs11208257 and a range of pathological conditions using a relatively large number of subjects. Full article

Background/Objectives: Personality traits influence motivation, self-regulation, and adaptation in high-performance sports, and are partially modulated by dopaminergic genetic variability. This study aimed to examine the association between the DRD2 Ex8 rs6276 polymorphism and NEO Five-Factor Inventory (NEO-FFI) personality traits in elite athletes and non-athlete controls. Methods: A total of 323 participants were included: 141 athletes and 182 controls. Genomic DNA was isolated from venous blood, and DRD2 Ex8 rs6276 genotypes (A/A, A/G, G/G) were determined using real-time PCR with melting-curve analysis. Personality traits were assessed using the NEO-FFI, and group differences as well as genotype × group interactions were evaluated using multivariate analyses and non-parametric tests. Results: Athletes scored significantly higher on Conscientiousness than controls. A genotype × group interaction was observed for Extraversion, and the main effect of the genotype was found to be Agreeableness. Athletes with the A/A genotype exhibited the highest Extraversion scores, whereas those with the G/G genotype demonstrated higher Agreeableness than other genotypes. Conclusions: These findings indicate that dopaminergic variation contributes to individual differences in social and motivational traits, which may support athletic engagement and adaptation to high-demand environments. The results should be interpreted with caution due to the moderate sample size, deviation from the Hardy–Weinberg equilibrium in the athlete group, and reliance on a single personality assessment tool. Full article